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An optimized method for measurement of gamma-H2AX in blood mononuclear and cultured cells

Nature Protocols volume 3, pages 1187–1193 (2008)Cite this article

Abstract

Phosphorylation of histone protein H2AX on serine 139 (gamma-H2AX) occurs at sites flanking DNA double-stranded breaks (DSBs) and can provide a measure of the number of DSBs within a cell. We describe a flow cytometry-based method optimized to measure gamma-H2AX in nonfixed mononuclear blood cells as well as in cultured cells, which is more sensitive and involves less steps compared with protocols involving fixed cells. This method can be used to monitor induction of gamma-H2AX in mononuclear cells from cancer patients undergoing radiotherapy and for detection of gamma-H2AX throughout the cell cycle in cultured cells. The method is based on the fact that H2AX like other histone proteins are retained in the nucleus when cells are lysed at physiological salt concentrations. Cells are therefore added without fixation to a solution containing detergent to lyse the cells along with a fluorescein isothiocyanate-labeled monoclonal gamma-H2AX antibody, DNA staining dye and blocking agents. The stained nuclei can be analyzed by flow cytometry to monitor the level of gamma-H2AX to determine the level of DSBs and DNA content and to determine the cell cycle stage. The omission of fixation simplifies staining and enhances the sensitivity. This protocol can be completed within 4–6 h.

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Figure 1: PFA-fixation results in lower gamma-H2AX signal.
Figure 2: Effect of different staining time.
Figure 3: Gamma-H2AX in different cell cycle stages upon etoposide treatment.
Figure 4: Gamma-H2AX measurements in patients irradiated with 5 Gy of IR to the pelvic area.

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Authors and Affiliations

  1. Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg University, Göteborg, SE-413 45, Sweden

    Aida Muslimovic, Yue Gao & Ola Hammarsten

  2. Department of Oncology, Cross Cancer Institute, University of Alberta, 11560 University Avenue, Edmonton, T6G 1Z2, Alberta, Canada

    Ismail Hassan Ismail

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  1. Aida Muslimovic
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  2. Ismail Hassan Ismail
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  3. Yue Gao
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  4. Ola Hammarsten
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Correspondence to Ola Hammarsten.

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Muslimovic, A., Ismail, I., Gao, Y. et al. An optimized method for measurement of gamma-H2AX in blood mononuclear and cultured cells. Nat Protoc 3, 1187–1193 (2008). https://doi.org/10.1038/nprot.2008.93

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